Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Solanum tuberosum tissues (including leaves, flowers and stolon). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the genome under study.
Project description:Phloem localization of plant viruses is advantageous for acquisition by sap-sucking vectors but hampers host-virus protein interaction studies. In this study, Potato leafroll virus (PLRV)-host protein complexes were isolated from systemically infected potato, a natural host of the virus. Comparing two different co-immunoprecipitation support matrices coupled to mass spectrometry, we identified 44 potato proteins and one viral protein (P1) specifically associated with virus isolated from infected phloem. An additional 142 proteins interact in complex with virus at varying degrees of confidence. Greater than 80% of these proteins were previously found to form high confidence interactions with PLRV isolated from the model host Nicotiana benthamiana. Bioinformatics revealed that these proteins are enriched for functions related to plasmodesmata, organelle membrane transport, translation and mRNA processing. Our results show that model system proteomics experiments are extremely valuable for understanding protein interactions regulating infection in recalcitrant pathogens such as phloem-limited viruses.
Project description:RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and non-human RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and non-human transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2 bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs. Characterization of non-coding and pathogen RNA-protein interactions using an automated computational pipeline and improved iCLIP biochemistry
Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Solanum tuberosum tissues (including leaves, flowers and stolon). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the genome under study. Small RNA libraries were derived from leaves, flowers and stolon of Solanum tuberosum. Total RNA was isolated using the Plant RNA Purification Reagent (Invitrogen), and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Barbara Baker for providing the plant material as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.
Project description:Solanum lycopersicum and Solanum tuberosum are agriculturally important crop species as they are rich sources of starch, protein, antioxidants, lycopene, beta-carotene, vitamin C, and fiber. The genomes of S. lycopersicum and S. tuberosum are currently available. However the linear strings of nucleotides that together comprise a genome sequence are of limited significance by themselves. Computational and bioinformatics approaches can be used to exploit the genomes for fundamental research for improving their varieties. The comparative genome analysis, Pfam analysis of predicted reviewed paralogous proteins was performed. It was found that S. lycopersicum proteins belong to more families, domains and clans in comparison with S. tuberosum. It was also found that mostly intergenic regions are conserved in two genomes followed by exons, intron and UTR. This can be exploited to predict regions between genomes that are similar to each other and to study the evolutionary relationship between two genomes, leading towards the development of disease resistance, stress tolerance and improved varieties of tomato.
Project description:Dickeya solani is a Gram-negative bacterium able to cause disease symptoms on a variety of crop and ornamental plants worldwide. Weeds including Solanum dulcamara (bittersweet nightshade) growing near agricultural fields have been reported to support populations of soft rot bacteria in natural settings. However, little is known about the specific interaction of D. solani with such weed plants that may contribute to its success as an agricultural pathogen. The aim of this work was to assess the interaction of D. solani with its crop plant (Solanum tuberosum) and an alternative (S. dulcamara) host plant. From a collection of 10,000 Tn5 transposon mutants of D. solani IPO2222 carrying an inducible, promotorless gusA reporter gene, 210 were identified that exhibited plant tissue-dependent expression of the gene/operon into which the Tn5 insertion had occurred. Thirteen Tn5 mutants exhibiting the greatest plant tissue induction of such transcriptional units in S. tuberosum or S. dulcamara as measured by qRT-PCR were assessed for plant host colonization, virulence, and ability to macerate plant tissue, as well as phenotypes likely to contribute to the ecological fitness of D. solani, including growth rate, carbon and nitrogen source utilization, motility, chemotaxis toward plant extracts, biofilm formation, growth under anaerobic conditions and quorum sensing. These 13 transcriptional units encode proteins involved in bacterial interactions with plants, with functions linked to cell envelope structure, chemotaxis and carbon metabolism. The selected 13 genes/operons were differentially expressed in, and thus contributed preferentially to D. solani fitness in potato and/or S. dulcamara stem, leaf, and root tissues.
Project description:Identification and characterization of potato long noncoding RNAs responsive to Pectobacterium carotovorum subspecies brasiliense infection [Time-course RNAseq]